Many commercially important analytical methods are based on the principle that the analytes can be recognised and quantified from a matrix with using label substances. For instance, in the assays based on the biological properties of analytes, such as in immunoassays, the analyte (A) can be selectively captured from a solution upon a solid support with the aid of antibodies immobilised on the surface of the solid support, and the amount of (A) can be quantified using another antibody selectively binding with (A) and being labelled with a suitable marker substance. Such a marker substance can be, for instance, radioactive isotope, enzyme, molecule that absorbs light or produces fluorescence or phosphorescence, certain metal chelates etc., which can be coupled with chemical bonds with an antibody. Alternatively, purified (A) can be labelled (A-L) and the amount of unlabelled (A) can be determined by antibodies immobilised on a solid support by exploiting competitive reaction between (A-L) and analyte (A). DNA- and RNA-probing assays are based on the analogous bioaffinity principles as immunoassays and can be performed along with related procedures. Also, other chemical and biochemical analysis methods can be based on analogous principles. Presently, there is an increasing need for multiparameter assays due to a growing demand to decrease the costs and/or increase the simplicity and accuracy of determinations. One solution to these problems is the use of label compounds luminescing at different wavelengths. Various methods and strategies in immunoassays are described, e.g., in "The Immunoassay Handbook", Edited by David Wild, Stockton Press Ltd., New York, 1994, pages 1-618.
It is already known that organic luminophores and metal chelates suitable for labelling in analytical methods can be excited with light or by electrochemical means resulting in the specific emission from the labelling substance. The methods based on these phenomena are generally sensitive and well-suited for the excitation of label substances. However, difficulties are encountered when the concentrations of labels in real assays are very low; e.g., the use of fluorescence is complicated by the existence of Tyndall, Raleigh, and Raman scattering, and by the background fluorescence common in biological samples. Phosphorescence in liquid phase is mainly usable only in connection with some specially synthesised lanthanide chelates. Utilisation of the long-lived photoluminescence of these compounds is restricted mainly due to complicated apparatus required and high cost of pulsed light sources.
Electrochemiluminescence can be generated in non-aqueous solvents at inert metal electrodes with a rather simple apparatus. However, bioaffinity assays which are of commercial importance are normally applicable in aqueous solutions only. Samples are practically always aqueous and therefore the detection method of a label substance must be applicable in aqueous solution. Presently, only certain transition metal chelates can serve as electrochemiluminescent labels in micellar solutions, which, in principle, are not fully aqueous solutions. However, these methods utilising conventional electrochemistry and insert metal electrodes do not allow simultaneous excitation of several label substances possessing sufficiently differing emission spectra and/or luminescence lifetime.
Mainly inert active metal (e.g. Pt and Au) or carbon electrodes are applied in conventional electrochemistry. Their utilisation is restricted to a narrow potential window due to the water decomposition reactions, hydrogen and oxygen evolution. Luminophores usable as fluorescent or phosphorescent labels cannot normally be electrically excited in aqueous solution at these electrodes due to the inaccessibility of the highly anodic and cathodic potentials required for the excitation reactions. With suitably selected semiconductor electrodes a wider potential window is achievable, but only very rare labelling substances can be excited at this type of electrodes in fully aqueous solutions.
The present invention provides considerable improvement for use of active metal electrodes or semiconductor electrodes and makes it possible to simultaneously excite a variety of different labelling substances in fully aqueous solution. The invention utilises a new type of electrodes, conductors covered with an insulating film, which are useless in the field of conventional electrochemistry. Below these electrodes are called either as insulator electrodes or insulating film-coated electrodes.